Hydrogels for soft tissue filling

Abstract

The invention relates to hydrogels that can retain their initial thickness over time. The cross-linked polysaccharide-based hydrogels have a prediction index PIdx greater than or equal to 80%.

Description

[0001] DESCRIPTION

[0002] TITLE: HYDROGELS FOR SOFT TISSUE FILLING

[0003] TECHNICAL FIELD

[0004] The present invention relates to filler gels, in particular based on crosslinked polysaccharides, used in the aesthetic and / or medical field to fill wrinkles and / or give more relief to certain areas of the face.

[0005] PRIOR TECHNIQUE

[0006] Filler gels are, for example, polysaccharide-based hydrogels, particularly hyaluronic acid (HA), and are injected under the skin using a syringe. Hydrogels suitable for soft tissue filling are typically crosslinked hydrogels, i.e., the polysaccharide is crosslinked using one or more crosslinking agents. This crosslinking results in a hydrogel with the mechanical properties desirable for soft tissue filling.

[0007] Manufacturers offer filler hydrogels with various rheological properties depending on the hydrogel injection site. For example, when injected into the superficial layers of the skin, hydrogels should lightly fill fine lines and wrinkles and be able to easily follow facial movements. Whereas hydrogels intended for mid- or deep implantation, particularly intended for filling more severe folds and wrinkles and / or creating volume, so-called "volumizing" hydrogels, should advantageously have an intrinsic capacity to sustainably maintain their thickness in the skin layers, including under the stress of facial movements.

[0008] The most commonly used rheological measurements of hydrogels are those of the moduli G' and G”, the phase shift angle 5 being related to these measurements (tan 5 = G” / G'). These measurements are commonly performed under low oscillatory stress with low amplitude, in their linear viscoelasticity zone. These measurements do not necessarily reflect the mechanical stress and deformation that a filling hydrogel undergoes in vivo. These measurements do not allow predicting the compressive behavior of the hydrogel. However, in the case of implantation, particularly deep implantation, the hydrogel is compressed between layers of tissue and its ability to create relief depends on its ability not to spread or lose its thickness excessively over time.It is known that commercial so-called "volumizing" hydrogels, after their in vivo application, tend not to effectively maintain their full thickness under the stress of surrounding tissues and / or facial movements. There is therefore an interest in developing volumizing hydrogels capable of sustainably maintaining their thickness in the skin layers, including under the stress of facial movements.

[0009] Furthermore, crosslinked hydrogels may present biocompatibility problems. To optimize this biocompatibility, one solution is to reduce the degree of polysaccharide modification required to obtain the gel in order to be as close as possible to the natural polysaccharide or to opt for alternative crosslinking methods, such as sol-gel crosslinking. However, with such solutions, the prepared hydrogels may not have the mechanical properties suitable for use as volumizers, in particular these hydrogels are likely to be less crosslinked, may have a reduced G' modulus and a poor capacity to maintain their thickness in tissues.A need therefore remains for the provision of hydrogels, in particular volumizing hydrogels, having a minimal degree of modification of the polysaccharide and nevertheless having mechanical properties suitable for the creation of volume and sustainably maintaining their thickness in the layers of the skin, including under the stress of facial movements. The present invention thus aims to provide hydrogels based on crosslinked polysaccharides which have an improved in vivo volumizing capacity, that is to say an ability to sustainably maintain their thickness in the layers of the skin, including under the stress of facial movements. The hydrogels proposed will advantageously be biocompatible, as natural and as little modified as possible.

[0010] SUMMARY OF THE INVENTION

[0011] The invention provides hydrogels based on crosslinked polysaccharide having a projection index PIdx greater than or equal to 80%, the projection index being determined according to the method described below in detail.

[0012] The invention also relates to a crosslinked polysaccharide-based hydrogel having a projection index PIdx greater than or equal to 80% prepared by a method comprising the following steps: a) providing at least one polysaccharide or a salt thereof; b) providing at least one crosslinking agent or a salt thereof; c) preparing a crosslinking reaction medium comprising the polysaccharide(s), the crosslinking agent(s) and a solvent, the total amount of crosslinking agent ranging from 0.001 to 0.03 mole, preferably from 0.001 to 0.02 mole per 1 mole of repeating unit of the polysaccharide, preferably the duration of the step of preparing the reaction medium not exceeding 5 hours;and d) placing the reaction medium obtained at the end of step c), at a pressure P less than or equal to atmospheric pressure and at a temperature T greater than the temperature of the eutectic point of the reaction medium as measured at pressure P and less than the temperature of the freezing point of the reaction medium as measured at pressure;

[0013] P, preferably for a period of 1 week to 17 weeks.

[0014] The invention also relates to a crosslinked polysaccharide-based hydrogel having a projection index PIdx greater than or equal to 80% prepared by a method comprising the following steps: a) providing at least one polysaccharide or a salt thereof; b) providing at least one functionalizing agent or a salt thereof; c) preparing a crosslinking reaction medium comprising the polysaccharide(s), the functionalizing agent(s) and a solvent, the total amount of functionalizing agent ranging from 0.001 to 0.50 mole, preferably from 0.005 to 0.45 mole, in particular from 0.10 to 0.25 mole per 1 mole of repeating unit of the polysaccharide, or ranging from 0.001 to 0.03 mole, preferably from 0.001 to 0.02 mole, per 1 mole of repeating unit of the polysaccharide, preferably the duration of the step of preparing the reaction medium not exceeding 5 hours;and d) placing the reaction medium obtained at the end of step c), at a pressure P less than or equal to atmospheric pressure and at a temperature T greater than the temperature of the eutectic point of the reaction medium as measured at pressure P and less than the temperature of the freezing point of the reaction medium as measured at pressure P, preferably for a period ranging from 1 week to 17 weeks.;

[0015] The invention also relates to the use of the hydrogels of the invention for preventing and / or treating alterations in the viscoelastic or biomechanical properties of the skin; for filling wrinkles, fine lines and scars; for reducing nasolabial folds and bitterness lines; for increasing the volume of the cheekbones, chin or lips or for reducing the appearance of wrinkles and fine lines.

[0016] Other aspects of the invention are as described in the claims and hereinafter. Definitions

[0017] The term "gel" refers to a polymer network that is expanded throughout its volume by a fluid. This means that a gel is formed of two media, one "solid" and one "liquid," dispersed in each other. The so-called "solid" medium consists of long polymer molecules connected to each other by weak bonds (e.g., hydrogen bonds) or by covalent bonds (crosslinking). The liquid medium consists of a solvent. A gel generally corresponds to a product that has a phase angle 5 less than or equal to 45° at 1 Hz for a strain of 0.1% or a pressure of 1 Pa, advantageously a phase angle 5 ranging from 2° to 45° or ranging from 20° to 45°.

[0018] The term "hydrogel" designates a gel as defined above in which the solvent constituting the liquid medium is predominantly water (for example at least 90%, in particular at least 95%, in particular at least 97%, in particular at least 98% by weight of the liquid medium). Preferably, the liquid medium comprises, in particular consists of, a buffer solution, advantageously allowing a pH of the liquid medium of between 6.8 and 7.8, in particular a saline phosphate buffer.

[0019] The term “injectable gel” refers to a gel that can flow and be injected manually using a syringe equipped with a needle with a diameter ranging from 0.1 to 0.5 mm, for example a 32G, 30G, 27G, 26G, 25G hypodermic needle. Preferably, an “injectable gel” is a gel having an average extrusion force of less than or equal to 25N, preferably ranging from 5 to 25N, more preferably ranging from 8 to 15N, when measured with a dynamometer, at a fixed speed of approximately 12.5 mm / min, in syringes with an external diameter greater than or equal to 6.3 mm, with a needle with an external diameter less than or equal to 0.4 mm (27G) and a length of 1”, at room temperature.

[0020] A “superficial application” means the administration, for example by mesotherapy, of a composition superficially into the skin, or onto the skin, for the treatment of the superficial layers of the skin, the epidermis and the most superficial parts of the dermis, to reduce superficial wrinkles and / or improve the quality of the skin (such as its radiance, density or structure) and / or rejuvenate the skin.

[0021] A “midline application” means the administration of a composition into the midline of the skin to treat the midline layers of the skin, as well as to reduce midline wrinkles.

[0022] A “deep application” refers to the administration of a hydrogel into the deepest layers of the skin, the hypodermis and the deepest part of the dermis, and / or beneath the skin (above the periosteum) to “volumize” soft tissues, such as for filling the deepest wrinkles and / or partially atrophied regions of the facial and / or body contour. So-called “volumizing” hydrogels can typically be administered for deep application.

[0023] A "crosslinked polysaccharide" refers to a polysaccharide modified during a crosslinking reaction. Crosslinking may be achieved by means of a crosslinking agent or result from a sol-gel reaction. When the crosslinking reaction is a sol-gel reaction, the polysaccharide is modified with a functionalizing agent.

[0024] Conversely, a “non-crosslinked polysaccharide” designates a polysaccharide not modified with a crosslinking agent and / or functionalizing agent and which therefore has not undergone a crosslinking reaction.

[0025] The term "crosslinking agent" refers to any compound capable of introducing crosslinking between different polysaccharide chains.

[0026] The term “functionalizing agent” refers to any compound capable of reacting with a functional group of the polysaccharide and reacting via a sol-gel reaction.

[0027] The “molar crosslinking rate” (TR), expressed in %, designates the molar ratio of the quantity of crosslinking agent to the quantity of repeating unit of the polysaccharide introduced into the crosslinking reaction medium expressed per 100 moles of repeating units of the polysaccharide in the crosslinking medium.

[0028] The “molar functionalization rate”, expressed in %, designates the molar ratio of the quantity of functionalizing agent relative to the quantity of repeating unit of the polysaccharide introduced into the crosslinking reaction medium expressed per 100 moles of repeating units of the polysaccharide in the functionalization medium.

[0029] The term "repeating unit" of a polysaccharide refers to a structural unit consisting of one or more (usually 1 or 2) monosaccharides whose repetition produces the complete polysaccharide chain.

[0030] The “degree of modification” (MOD) of a polysaccharide, such as hyaluronic acid, corresponds to the molar quantity of crosslinking agent bound to the polysaccharide and / or functionalizing agent bound to the polysaccharide, by one or more of its ends, expressed per 100 moles of repeating units of the polysaccharide. It can be determined by methods known to those skilled in the art such as Nuclear Magnetic Resonance (NMR) spectroscopy (see experimental section).

[0031] The term "polysaccharide" refers to a polymer composed of monosaccharides (preferably D-enantiomers) joined together by glycosidic bonds. "Room temperature" means a temperature ranging from 20 to 25°C, more particularly 25°C.

[0032] DETAILED DESCRIPTION OF THE INVENTION

[0033] The proposed crosslinked polysaccharide-based hydrogels are characterized by a projection index Pldx greater than or equal to 80%, the projection index being determined as described in detail below. The projection index reflects the rheological behavior of the hydrogels under force over time. Hydrogels with a projection index Pldx greater than or equal to 80% have the ability to maintain their initial thickness over time. In other words, such hydrogels have the ability to maintain a thickness greater than or equal to 80% of their initial thickness over time. They are therefore particularly suitable for use as "volumizing hydrogels".

[0034] The projection index therefore makes it possible to discriminate between hydrogels, to select the one or ones with the most interesting properties for the desired result, and in particular, when seeking to obtain so-called “volumizing” hydrogels.

[0035] Determination of the projection index

[0036] The projection index is determined by a method comprising the following steps:

[0037] 1) subjecting a bolus of one gram of hydrogel to a constant fixed compression force F of 2 newtons, said hydrogel being previously placed between two circular pressure plates of a rheometer and having an initial thickness dO of 700 pm given by the distance between the two plates, the two plates being parallel and having a diameter of 40 mm,

[0038] 2) acquire at 25°C the evolution of the variation of the thickness of the hydrogel thus compressed for 1 hour;

[0039] 3) parameterize a generalized Maxwell model approximating the evolution observed from the acquisition carried out in step 2), said model expressing the thickness of the hydrogel as a function of time d gei(t) and corresponding to the following equation 1: dgel(t) = dœ + Z Ai.e<^

[0040] (equation 1) where d~ is the limit thickness obtained at equilibrium, Ai a constant, Ti a relaxation parameter, the number of members i of the equation being equal to 2;

[0041] 4) determine from the model a limit thickness d~ towards which the hydrogel tends to evolve over time; and 5) generate the projection index PIdx from the following equation 2:

[0042] (equation 2).

[0043] The limit thickness obtained at equilibrium (d~) designates the thickness obtained after an infinite time for the applied compression force F.

[0044] The applied compression force F (2N) is preferably within the linear viscoelastic deformation range of the hydrogel, i.e. the applied compression force F advantageously translates into a pressure within the linear viscoelastic range of the hydrogel. Such a force value is suitable for so-called volumizing filling gels. The linear viscoelastic region (Linear ViscoElastic Region or LVER) corresponds to the range of deformations of the hydrogel from an initial value of elastic modulus E' up to the value of the elastic modulus E' reduced by 10% of its initial value.

[0045] The method for determining the projection index can thus initially comprise a step of determining the linear viscoelastic deformation range of the hydrogel. This step makes it possible to ensure that the applied compression force F is indeed within the linear viscoelastic deformation range of the gel. Advantageously, this step consists of an oscillatory stress scanning measurement in compression mode at a given oscillation frequency to determine the linear viscoelastic region and frame the applied normal force. This measurement is applied over a stress range determined under conditions identical to the conditions for measuring the projection index. Preferably, the stress range covers from 10 Pa to 10,000 Pa at 1 Hz at 25°C, geometry of parallel flat plates (40mm diameter anodized aluminum, TA instruments®), the distance between the base plate and the pressure plate is equal to 700 μm.

[0046] When the applied compressive force F is in the linear viscoelastic range LVER of the hydrogels, the projection index PIdx is a direct measure of their ability to maintain their initial thickness under a given compressive stress. The projection index thus reflects the behavior of the hydrogel within the tissues.

[0047] When the applied compressive force F is beyond their linear viscoelasticity range LVER, the projection index PIdx gives an indication of their ability to maintain their thickness over a certain period; in theory, in an unconfined geometry, these hydrogels would continue to creep, but in reality, the surrounding tissues provide some confinement blocking the creep of the gel and thus PIdx remains an information of interest to predict the behavior of the gel in vivo. Preferably, the measurement of the projection index is carried out using a DHR2 rheometer equipped with a geometry of parallel flat plates (diameter 40mm anodized aluminum, TA instruments®) associated with the TRIOS software (TA Instruments®). More specifically, the determination of the projection index can be carried out as follows. A bolus (sample) of 1 gram of hydrogel is deposited on a base plate of the rheometer.A pressure plate with a diameter of 40 mm applies a force to the sample until its thickness, given by the distance between the base plate and the pressure plate, is equal to 700 pm (d0).

[0048] Once the thickness has reached this predefined value d0 and, preferably, the resistance force of the hydrogel is less than or equal to the value of the force F to be applied, a fixed and constant compression force F of 2N (Newton) is applied and the evolution of the variation of the thickness of the hydrogel is acquired. The acquisition begins by taking this instant as the origin of the acquisition times. This acquisition is carried out for 1 hour at 25°C. Preferably, the parameter d~ is calculated using the OriginPro® software version 9.6.0.172.

[0049] This software is used to apply a mathematical model in order to have the best fit of the model with the observed thickness evolution.

[0050] The model used is that of generalized Maxwell, which gives the evolution of the thickness of the hydrogel as a function of time t by the formula:

[0051] Good results are obtained from the presence of two exponential terms: dgei( d~ corresponds to the thickness at equilibrium after an infinite time, Ai a constant, Ti a relaxation parameter, the number of members i of the equation being equal to 2.

[0052] The OriginPro® software allows the values ​​of the parameters Ai and Ti to be calculated / determined by iteration. Ai and Ti reflect a physical phenomenon of relaxation of a hydrogel. These are positive values. In particular, Ai (equivalent to a thickness) is greater than 0 and less than or equal to 700 pm. In particular, Ti (equivalent to a time) is less than 24 hours, preferably less than 2 hours, more preferably less than 1 hour. In particular, the iteration is stopped when R2 >0.99.

[0053] The projection index values ​​are then calculated as follows: 100

[0054] The do corresponds to the initial thickness between the plates before application of force.

[0055] The parameters set for the application of the method for determining the projection index to characterize the hydrogel according to the invention allow for easy discrimination of the projection index values ​​while applying a compression force included in the LVER of the so-called “volumizing” hydrogels.

[0056] The crosslinked polysaccharide-based hydrogels of the present invention have a projection index greater than or equal to 80%, preferably greater than or equal to 82%, even more preferably greater than or equal to 85%, even more preferably greater than or equal to 90%. The projection index is determined by the method detailed above.

[0057] In certain embodiments, the hydrogel according to the invention has a projection index PIdx greater than or equal to 81%, preferably greater than or equal to 82%, more preferably greater than or equal to 83%, more preferably greater than or equal to 84%.

[0058] In certain embodiments, the hydrogel according to the invention has a projection index PIdx greater than or equal to 86%, preferably greater than or equal to 87%, more preferably greater than or equal to 88%, more preferably greater than or equal to 89%.

[0059] A hydrogel having a projection index greater than or equal to 80% has the ability to sustainably maintain its thickness in the layers of the skin, including under the stress of facial movements, compared to a hydrogel having a lower projection index. In particular, the hydrogel according to the invention is a volumizing hydrogel intended for filling severe folds and wrinkles and / or creating volume.

[0060] The hydrogels according to the invention are based on crosslinked polysaccharide. The hydrogels according to the present invention therefore comprise one or more crosslinked polysaccharides. Crosslinking makes it possible to obtain a hydrogel having desirable mechanical properties for filling soft tissues.

[0061] The polysaccharide may be any polymer composed of monosaccharides joined together by glycosidic bonds or mixtures thereof. Preferably, the polysaccharide is chosen from pectin and pectic substances; chitosan; chitin; cellulose and its derivatives; agarose; glycosaminoglycans such as hyaluronic acid, heparosan, dermatan sulfate, keratan sulfate, chondroitin and chondroitin sulfate; and mixtures thereof. Even more preferably, the polysaccharide is chosen from hyaluronic acid, heparosan, chondroitin and mixtures thereof, even more preferably the polysaccharide is hyaluronic acid or one of its salts, in particular a physiologically acceptable salt such as the sodium salt, the potassium salt, the zinc salt, the calcium salt, the magnesium salt, the silver salt, the calcium salt and mixtures thereof.More specifically, hyaluronic acid is in its acid form or in the form of sodium salt (NaHA). The hydrogel can thus be a hydrogel based on hyaluronic acid and / or one of its salts.

[0062] The polysaccharide generally has a weight average molecular mass ranging from 0.03 to 10 MDa.

[0063] Preferably, if the polysaccharide is hyaluronic acid or one of its salts, it has a weight average molecular mass (Mw) ranging from 0.05 to 10 MDa, preferably ranging from 0.5 to 5 MDa, for example ranging from 0.07 to 10 MDa or from 0.07 to 5 MDa, or from 0.5 to 5 MDa or from 1 to 5 MDa or from 2 to 4 MDa.

[0064] Crosslinking agents

[0065] The polysaccharide may be crosslinked by means of a crosslinking agent, preferably selected from epoxy or non-epoxy bi- or multifunctional crosslinking agents, i.e. prepared by reaction of the polysaccharide with a crosslinking agent as described. Among the epoxy agents, mention may be made of 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxyoctane, 1,2-bis(2,3-epoxypropyl)-2,3-ethane (EGDGE), poly(ethylene glycol)-diglycidyl ether (PEGDG), and mixtures thereof. Among the non-epoxy agents that may be mentioned are endogenous polyamines such as spermine, spermidine and putrescine, aldehydes such as glutaraldehyde, carbodiimides and divinylsulfone, hydrazide derivatives such as adipic acid dihydrazide, bisalkoxyamine, dithiols such as polyethylene glycol dithiol and their mixtures.Among the non-epoxy agents, mention may be made of amino acids such as cysteine, lysine; peptides or proteins containing amino acids such as cysteine, lysine; trimetaphosphates, such as sodium trimetaphosphate, calcium trimetaphosphate, or barium trimetaphosphate.

[0066] In some embodiments, the crosslinking agent is an epoxy agent, preferably 1,4-butanediol diglycidyl ether (BDDE) or polyethylene glycol diglycidyl ether. Preferably the crosslinking agent is 1,4-butanediol diglycidyl ether (BDDE).

[0067] In certain embodiments, the crosslinking agent is a non-epoxy agent, preferably selected from endogenous polyamines, aldehydes, carbodiimides, divinylsulfone, amino acids, peptides and mixtures thereof. The hydrogel according to the present invention is preferably based on a crosslinked polysaccharide whose molar crosslinking rate is less than or equal to 3%. More preferably, the hydrogel according to the present invention is based on a crosslinked polysaccharide whose molar crosslinking rate is greater than 0 and less than or equal to 2%, preferably less than or equal to 1.5%, more preferably less than or equal to 1%, even more preferably less than or equal to 0.8% or 0.5%, in particular ranging from 0.1% to 0.8% or from 0.1% to 0.5% (number of moles of crosslinking agent(s) per 100 moles of repeating unit of the polysaccharide(s).

[0068] Functionalizing agent

[0069] The polysaccharide may be crosslinked using a functionalizing agent. The functionalizing agent enables crosslinking of the polysaccharide by sol-gel reaction. Thus, the functionalizing agent typically comprises a single function capable of reacting with a functional group of the polysaccharide and comprises a silylated group capable of reacting with another silylated group via a sol-gel reaction so as to enable crosslinking of the polysaccharide and form a hydrogel.

[0070] The functionalizing agent is typically a molecule with the formula Chem. It as presented below:

[0071] Chem. It or a salt thereof, in which:

[0072] T represents an isocyanate, amino, epoxide, carboxyl, N-succinimidyloxycarbonyl, N-sulfosuccinimidyloxycarbonyl, halocarbonyl, isothiocyanate, vinyl, formyl, hydroxyl, sulfhydryl, hydrazino, acylhydrazino, aminoxy, carbodiimide group, or an acid anhydride residue;

[0073] A represents a chemical bond or a spacer group;

[0074] R5 and R6, which may be identical or different, represent a hydrogen atom; a halogen atom; a group -OR4 with R4 representing a hydrogen atom, an aryl group or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms; an aryl; or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms optionally substituted by one or more group(s) chosen from a halogen atom, an aryl and a hydroxyl; R10 represents a hydrogen atom, an aryl group or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms.

[0075] Preferably, in the formula Chem. II, T represents an isocyanate, sulfhydryl, amino, epoxide, vinyl, formyl, or carbodiimide group, more preferably, T represents an epoxide or amino group, even more preferably T represents an epoxide group.

[0076] Preferably, in the formula Chem. II, A represents a spacer group, more preferably a divalent aliphatic hydrocarbon chain, in particular linear or branched and saturated, comprising from 1 to 12 carbon atoms:

[0077] - in which are optionally intercalated, between two carbon atoms of said chain, one or more (in particular 1, 2, 3 or 4) divalent units chosen from arylenes, -O-, -S-, -S(O)-, -C(=O)-, -SO2- and -N(R9)- with R9 representing a hydrogen atom, an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms, or an aryl-(C1-C6)alkyl,

[0078] - said chain being unsubstituted or substituted by one or more monovalent groups chosen from a halogen atom, a hydroxyl, an aryl-(C1-C6)alkyl.

[0079] Advantageously, A is a divalent aliphatic hydrocarbon chain, in particular linear or branched and saturated, in which are optionally intercalated, between two carbon atoms of said chain, one or more divalent units -O-, more advantageously from 1 to 4 divalent units -O-, even more advantageously a divalent unit O.

[0080] Preferably, A is a (C1-C12)alkylene chain in which are optionally intercalated, between two carbon atoms of said chain, one or more divalent -O- units, more preferably from 1 to 4 divalent -O- units, even more preferably one divalent -O- unit.

[0081] In particular, A represents a divalent chain -(C1-C6)alkylene-O-(C1-C6)alkylene-, in particular -(C1-C4)alkylene-O-(C1-C4)alkylene-, more particularly a divalent chain -CH2-O-(CH2)3-, the CH2 group being linked to T and the (CH2)3 group being linked to Si in the molecule of formula Chem. II.

[0082] Advantageously, the spacer group will also avoid steric hindrance between the silylated group and the T group of the molecule of formula Chem. Il, while ensuring a stable bond between these two groups.

[0083] Preferably, in the formula Chem. II, R5 and R6, identical or different, represent a group -OR4 with R4 representing a hydrogen atom, an aryl group or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms; or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms optionally substituted by one or more groups chosen from a halogen atom, an aryl and a hydroxyl.

[0084] In particular, R5 and R6, identical or different, represent a -OR4 group with R4 representing a (C1-C6)alkyl group; or a (C1-C6)alkyl group.

[0085] Advantageously, R5 and R6, identical or different, represent a group -OR4 with R4 representing a hydrogen atom, an aryl group or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms, preferably with R4 representing an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms, such as a (C1-C6)alkyl group.

[0086] Preferably, in the formula Chem. II, R10 represents a hydrogen atom or an aliphatic hydrocarbon group having from 1 to 6 carbon atoms such as a (C1-C6)alkyl group, more preferably R10 represents an aliphatic hydrocarbon group having from 1 to 6 carbon atoms such as a (C1-C6)alkyl group.

[0087] Preferably, the molecule of formula Chem. It is such that:

[0088] - T is as defined above and advantageously represents an amino or epoxide group, preferably an epoxide group;

[0089] - A is a divalent chain -(C1-C6)alkylene-O-((C1-C6)alkylene-, in particular -(C1-C4)alkylene-O-(C1-C4)alkylene-, such as -CH2-O-(CH2)3-, the CH2 group preferably being linked to T and the (CH2)3 group being linked to Si in the molecule of formula Chem. II;

[0090] - R5 and R6, identical or different, are each a -OR4 group with R4 representing a (C1-C6)alkyl group, preferably a methyl or an ethyl; or a (C1-C6)alkyl group, preferably a methyl or an ethyl; and

[0091] - R10 is a (C1-C6)alkyl group, preferably methyl or ethyl; the groups R5, R6 and OR10 may be identical.

[0092] In particular, the molecule of formula Chem. It is chosen from (3-aminopropyl)triethoxysilane (APTES), (3-glycidyloxypropyl)trimethoxysilane (GPTMS), 3- Glycidoxypropyldimethoxymethylsilane, 3-Glycidoxypropyldimethylethoxysilane, (3-glycidyloxypropyl)ethoxydimethoxysilane, (3-glycidyloxypropyl)triethoxysilane, diethoxy(3-glycidyloxypropyl)methylsilane, and mixtures thereof; preferably from (3-glycidyloxypropyl)trimethoxysilane (GPTMS), (3-glycidyloxypropyl)ethoxydimethoxysilane, (3-glycidyloxypropyl)triethoxysilane, diethoxy(3-glycidyloxypropyl)methylsilane, and mixtures thereof; even more preferably (3-aminopropyl)triethoxysilane (APTES), (3-glycidyloxypropyl)trimethoxysilane (GPTMS) and mixtures thereof. The hydrogel according to the present invention is preferably based on a crosslinked polysaccharide whose molar functionalization rate is less than 50%.More preferably, the hydrogel according to the present invention is based on a crosslinked polysaccharide whose molar functionalization rate ranges from 1 to 50% or from 5 to 45% or from 10 to 25% (number of moles of functionalizing agent(s) per 100 moles of repeating unit of the polysaccharide(s)). In certain embodiments, the hydrogel according to the present invention is based on a crosslinked polysaccharide whose molar functionalization rate ranges from 0.1 to 3% or from 0.1 to 2%.

[0093] The hydrogel according to the present invention is preferably based on a crosslinked polysaccharide having a degree of modification (MOD) of less than or equal to 3%, preferably less than or equal to 2%, more preferably less than or equal to 1%.

[0094] Advantageously, the hydrogel according to the present invention is based on a polysaccharide having a degree of modification (MOD) less than or equal to 1.8%, more preferably less than or equal to 1.5%, preferably less than or equal to 1.2%, even more preferably less than 1%.

[0095] The hydrogel according to the present invention advantageously has a degree of modification (MOD) / PIdx ratio of less than or equal to 0.050, preferably less than or equal to 0.040, more preferably less than or equal to 0.030, even more preferably less than or equal to 0.015. Advantageously, the hydrogel according to the present invention has a degree of modification (MOD) / PIdx ratio of less than or equal to 0.038, preferably less than or equal to 0.025, more preferably less than or equal to 0.020, even more preferably less than or equal to 0.012.

[0096] The hydrogel according to the present invention may comprise from 0.1 to 5% by weight, preferably of

[0097] 1 to 3% by weight of polysaccharide (e.g. hyaluronic acid), relative to the total weight of the hydrogel, the polysaccharide (e.g. hyaluronic acid) being present in crosslinked form. The hydrogel may further comprise a polysaccharide in non-crosslinked form. In particular, the content of non-crosslinked polysaccharide (e.g. hyaluronic acid) may vary from 0.5 to 40% by weight, preferably from 1 to 40% by weight, more preferably from

[0098] 2 to 30% by weight, more preferably 2 to 20% by weight, relative to the total weight of polysaccharide (e.g. hyaluronic acid) present in the hydrogel.

[0099] The total polysaccharide concentration in the hydrogel advantageously varies from 1 mg / g to 50 mg / g of hydrogel, more advantageously from 5 mg / g to 35 mg / g of hydrogel, even more advantageously from 10 mg / g to 30 mg / g of hydrogel. Preferably, this polysaccharide is hyaluronic acid, even more preferably sodium hyaluronate. The hydrogel according to the invention may comprise one or more polysaccharides having advantageously undergone a crosslinking step for a period ranging from 1 week to 17 weeks or from 2 weeks to 17 weeks at a pressure P less than or equal to atmospheric pressure and at a temperature T higher than the temperature of the eutectic point of the reaction medium as measured at pressure P and lower than the temperature of the freezing point of the reaction medium as measured at pressure P.

[0100] The pressure P is preferably atmospheric pressure. "Atmospheric pressure" is the pressure exerted by the air constituting the atmosphere on any surface in contact with it.

[0101] The temperature of the reaction medium (temperature T) is preferably greater than or equal to -55°C and less than or equal to -5°C, preferably it ranges from -35°C to -10°C. Even more preferably, the temperature T is approximately -20°C.

[0102] The hydrogel according to the present invention is preferably a sterile hydrogel, in particular heat sterilized, typically at a plateau temperature of 121°C to 135°C, preferably for a plateau time ranging from 1 minute to 20 minutes with FO > 15. The sterilizing value FO corresponds to the time required, in minutes, at 121°C, to inactivate 90% of the population of microorganisms present in the product to be sterilized. Alternatively, sterilization can be carried out by gamma radiation, UV radiation or by means of ethylene oxide.

[0103] The hydrogel has a physiological pH, i.e., ranging from 6.8 to 7.8. The pH of the hydrogel according to the present invention is preferably greater than or equal to 6.9 and less than or equal to 7.4; 7.3; 7.2; 7.1 or 7.

[0104] The hydrogel according to the present invention is preferably an injectable hydrogel, i.e. one which can flow and be injected manually by means of a syringe equipped with a needle with a diameter ranging from 0.1 to 0.5 mm, for example a 32G, 30G, 27G, 26G, 25G hypodermic needle.

[0105] In some embodiments, the hydrogel of the present invention further comprises an additional component selected from the group consisting of anesthetic agents, antioxidants, amino acids, peptides, proteins such as elastin, vitamins, minerals, nucleic acids, nucleotides, nucleosides, coenzymes, adrenergic derivatives, sodium dihydrogen phosphate monohydrate and / or dihydrate, sodium chloride and a mixture thereof. Non-crosslinked polysaccharides, in particular non-crosslinked hyaluronic acid, non-crosslinked heparosan or a mixture thereof, may be cited as an example of a lubricating agent.

[0106] Examples of anesthetics include, but are not limited to, Ambucaine, Amoxecaine, Amylein, Aprindine, Aptocaine, Articaine, Benzocaine, Betoxycaine, Bupivacaine, Butacaine, Butamben, Butanilicaine, Chlorobutanol, Chloroprocaine, Cinchocaine, Clodacaine, Cocaine, Cryofluorane, Cyclomethycaine, Dexivacaine, Diamocaine, Diperodon, Dyclonine, Etidocaine, Euprocine, Febuvérine, Fomocaine, Guafecainol, Heptacaine, Hexylcaine, Hydroxyprocaine, Hydroxytetracaine, Isobutamben, Leucinocaine, Levobupivacaine, Levoxadrol, Lidamidine, Lidocaine, Lotucaine, Menglytate, Mepivacaine, Meprylcaine, Myrtecaine, Octacaine, Octodrine, Oxetacaine, Oxybuprocaine, Parethoxycaine, Paridocaine, Phenacaine, Piperocaine, Piridocaine, Polidocanol, Pramocaine, Prilocaine, Procaine, Propanocaine, Propipocaine, Propoxycaine, Proxymetacaine,Pyrrocaine, Quatacaine, Quinisocaine, Risocaine, Rodocaine, Ropivacaine, Tetracaine, Tolycaine, Trimecaine, and one of their salts, in particular a hydrochloride salt, or a mixture thereof. Preferably, the hydrogel according to the invention comprises an anesthetic agent as defined above and in particular lidocaine, mepivacaine or one of their salts such as the hydrochloride. In certain embodiments, the hydrogel according to the invention comprises an anesthetic agent, in particular mepivacaine, lidocaine or one of their salts; more particularly a hydrochloride salt; preferably in amounts ranging from 0.1 to 30 mg / ml, for example from 0.5 to 10 mg / ml or more preferably from 2 to 5 mg / ml.,

[0107] Examples of antioxidants include, but are not limited to, glutathione, reduced glutathione, ellagic acid, spermine, resveratrol, retinol, L-carnitine, polyols, polyphenols, flavonols, theaflavins, catechins, caffeine, ubiquinol, ubiquinone, alpha-lipoic acid and their derivatives, and a mixture thereof.

[0108] Examples of amino acids include, but are not limited to, arginine (eg, L-arginine), isoleucine (eg, L-isoleucine), leucine (eg, L-leucine), lysine (eg, L-lysine or L-lysine monohydrate), glycine, valine (eg, L-valine), threonine (eg, L-threonine), proline (eg, L-proline), methionine, histidine, phenylalanine, tryptophan, cysteine, their derivatives (eg, N-acetylated derivatives such as N-acetyl-L-cysteine), and a mixture thereof.

[0109] Examples of vitamins and their salts include, but are not limited to, vitamins E, A, C, B, especially vitamins B6, B8, B4, B5, B9, B7, B12, and more preferably pyridoxine and its derivatives and / or salts, preferably pyridoxine hydrochloride. Examples of minerals include, but are not limited to, zinc salts (e.g., zinc acetate, in particular dehydrated, zinc citrate), magnesium salts, calcium salts (e.g., hydroxyapatite, in particular in bead form), potassium salts, manganese salts, sodium salts, copper salts (e.g., copper sulfate, in particular pentahydrate), optionally in a hydrated form, and mixtures thereof.

[0110] Examples of nucleic acids include, but are not limited to, adenosine, cytidine, guanosine, thymidine, cytodine, their derivatives, and a mixture thereof. As coenzymes, coenzyme Q10, CoA, NAD, NADP, and mixtures thereof may be cited.

[0111] Adrenaline derivatives include adrenaline, noradrenaline, and a mixture of these.

[0112] Hydrogel preparation process

[0113] The hydrogel according to the invention can be obtained by any preparation method known to those skilled in the art.

[0114] In particular, the hydrogel may be prepared by a method comprising the following steps: a) providing at least one polysaccharide or a salt thereof; b) providing at least one crosslinking agent or a salt thereof; c) preparing a crosslinking reaction medium comprising the polysaccharide(s), the crosslinking agent(s) and a solvent, the total amount of crosslinking agent ranging from

[0115] 0.001 to 0.03 mole per 1 mole of repeating unit of the polysaccharide, preferably the duration of the step of preparing the reaction medium not exceeding 5 hours; and d) crosslinking reaction: placing the reaction medium obtained at the end of step c) under conditions allowing the crosslinking of the polysaccharide by reaction of the polysaccharide with the crosslinking agent(s), namely at a pressure P less than or equal to atmospheric pressure and at a temperature T higher than the temperature of the eutectic point of the reaction medium as measured at pressure P and lower than the temperature of the freezing point of the reaction medium as measured at pressure

[0116] P, preferably for a period of 1 week to 17 weeks or 2 to 17 weeks.

[0117] In a variant of the process for preparing the hydrogel described above, the crosslinking agent is replaced by a functionalizing agent. In such a variant, the crosslinking reaction d) is carried out by sol-gel reaction of the functionalized polysaccharide, the functionalized polysaccharide being obtained by reaction of the polysaccharide with the functionalizing agent(s); the crosslinking reaction d) is carried out at a pressure P less than or equal to atmospheric pressure and at a temperature T greater than the temperature of the eutectic point of the reaction medium as measured at the pressure P and less than the temperature of the freezing point of the reaction medium as measured at the pressure P, preferably for a period ranging from 1 week to 17 weeks or from 2 to 17 weeks.In such a variant of the process for preparing the hydrogel, step b) comprises the provision of at least one functionalizing agent and step c) comprises the preparation of a reaction medium comprising the polysaccharide(s), the functionalizing agent(s) and a solvent, the total amount of functionalizing agent typically ranging from 0.001 to 0.50 per 1 mole of repeating unit of the polysaccharide.

[0118] Crosslinked polysaccharide hydrogels prepared by the methods described above are highly biocompatible. Indeed, such hydrogels are prepared with lower amounts of crosslinking agent. They nevertheless exhibit mechanical properties suitable for volume creation.

[0119] Furthermore, hydrogels prepared by such processes and having a Pldx projection index greater than or equal to 80% maintain their thickness durably in the layers of the skin, including under the stress of facial movements. Such hydrogels are particularly suitable for use as volumizing hydrogels.

[0120] Providing at least one polysaccharide or a salt thereof (step a))

[0121] Step a) of the process for preparing the hydrogel according to the invention comprises providing at least one polysaccharide or a salt thereof, in particular a physiologically acceptable salt thereof.

[0122] The polysaccharide is as described above. Preferably, the polysaccharide is hyaluronic acid or a salt of hyaluronic acid, preferably a sodium salt.

[0123] The polysaccharide may be provided in hydrated form, totally or partially, or in dry form, such as in powder or fiber form. More particularly, in step a), the polysaccharide is provided in dry form such as in powder or fiber form. When the polysaccharide is provided in hydrated form, it is in the form of a non-crosslinked gel or a solution. In particular, when the polysaccharide is in hydrated form, it is an aqueous non-crosslinked gel or an aqueous solution. More particularly, the polysaccharide is mixed with water, optionally with a phosphate buffer or a supplemented phosphate buffer, i.e. possibly comprising additional components as defined above. It should therefore be understood that the aqueous non-crosslinked gel or the aqueous polysaccharide solution does not comprise sodium hydroxide.

[0124] Provision of at least one crosslinking agent or salt thereof and / or functionalizing agent

[0125] Step b) of the process for preparing the hydrogel according to the invention comprises providing at least one crosslinking agent or a salt thereof, and / or at least one functionalizing agent, the functionalizing agent allowing crosslinking of the polysaccharide by sol-gel reaction. The crosslinking agent and the functionalizing agent are as described above.

[0126] Preparation of the crosslinking reaction medium (step c))

[0127] Step c) of the process for preparing the hydrogel according to the invention comprises the preparation of a crosslinking reaction medium. The reaction medium comprises the polysaccharide(s), the crosslinking agent(s) and / or functionalizing agent(s) and a solvent.

[0128] The solvent is typically water or a mixture comprising water and an organic solvent (typically a mixture comprising at least 90% by weight of water, or at least 95% or at least 98% by weight of water relative to the total weight of the solvent). For example, an organic solvent such as an alcohol, in particular ethanol, or DMSO, may be used to solubilize the crosslinking agent, for example when it is poly(dimethylsiloxane) terminated at each end by a diglycidyl ether (CAS number: 130167-23-6), before its addition to the aqueous reaction medium.

[0129] The reaction medium may further comprise salts, pH adjusters, for example a Bronsted base, more preferably a hydroxide salt, such as sodium or potassium hydroxide, additional components as described above and mixtures thereof. The addition of a Bronsted base may be particularly necessary when the functional groups of the crosslinking agent have an epoxide group or a vinyl group. In these cases, the crosslinking takes place at a pH greater than or equal to 10, more preferably greater than or equal to 12, which requires the addition of a Bronsted base to the reaction medium (for example sodium hydroxide), typically at a concentration of between 0.10M and 0.30M.

[0130] The total amount of crosslinking agent in the reaction medium is less than or equal to 0.03 mole or less than or equal to 0.02 mole, preferably less than or equal to 0.015 mole, even more preferably less than or equal to 0.01 mole per 1 mole of repeating unit of the polysaccharide. Preferably it varies from 0.001 to 0.03 mole per 1 mole of repeating unit of the polysaccharide, preferably from 0.001 to 0.02 mole or from 0.001 to 0.015 mole per 1 mole of repeating unit of the polysaccharide, more preferably from 0.001 to 0.01 mole per 1 mole of repeating unit of the polysaccharide, even more preferably from 0.001 to 0.008 or from 0.001 to 0.005 mole per 1 mole of repeating unit of the polysaccharide. When the polysaccharide is a glycosaminoglycan such as hyaluronic acid, the repeating unit is a disaccharide unit.

[0131] The total amount of functionalizing agent in the reaction medium typically varies from 0.001 to 0.50, preferably from 0.005 to 0.45, in particular from 0.10 to 0.25 mole per 1 mole of repeating unit of the polysaccharide or varying from 0.001 to 0.03 mole, preferably from 0.001 to 0.02 mole per 1 mole of repeating unit of the polysaccharide. For example, the total amount of formula Chem. It or a salt thereof in the reaction medium typically varies from 0.001 to 0.50, preferably from 0.005 to 0.45, in particular from 0.10 to 0.25 mole per 1 mole of repeating unit of the polysaccharide or varying from 0.001 to 0.03 mole, preferably from 0.001 to 0.02 mole per 1 mole of repeating unit of the polysaccharide.Typically, the higher the weight-average molecular mass Mw of the polysaccharide, the lower the functionalization rate will be in order to obtain a hydrogel having equivalent mechanical properties, in particular similar viscoelastic properties (in particular elastic modulus G', stress at the intersection of G' and G” and / or phase angle 5). In other words, the higher the weight-average molecular mass Mw of the polysaccharide, the lower the molar quantity of functionalizing agent, for example in molecule of formula Chem. II, in the reaction medium will be. The functionalization of the polysaccharide is typically carried out in an aqueous reaction medium.

[0132] The mass concentration of polysaccharide or polysaccharide salt in the reaction medium advantageously varies from 50 to 300 mg / g of solvent, preferably from 100 to 200 mg / g.

[0133] Step c) of the process typically comprises a step of homogenization of the reaction medium. Homogenization is generally carried out by three-dimensional stirring, stirring with a mixer, stirring with paddles or stirring with a spatula.

[0134] The reaction medium is typically prepared from the polysaccharide or polysaccharides in a dry form. When the reaction medium is prepared from the polysaccharide or polysaccharides in a hydrated form, the aqueous non-crosslinked gel or aqueous polysaccharide solution used for the preparation of the reaction medium does not include sodium hydroxide.

[0135] Step c) is typically carried out at a temperature ranging from 4 to 35°C, preferably 15°C to 25°C. Preferably, the duration of step c) does not exceed 5 hours. It generally varies from 15 minutes to 4 hours, preferably from 30 min to 2 hours. Thus, the contact time of the polysaccharide with sodium hydroxide before starting step d), whether the polysaccharide is provided in dry or hydrated form, generally does not exceed 5 hours, for example from 15 minutes to 4 hours or from 30 min to 2 hours. The reaction medium obtained at the end of step c) is advantageously placed directly under the conditions of step d) according to the invention.

[0136] Crosslinking at temperature T and pressure P (step d)

[0137] Step d) of crosslinking of the processes described above consists of crosslinking the polysaccharide(s). It consists of reacting / allowing the reaction medium to react to obtain a hydrogel based on crosslinked polysaccharide.

[0138] Crosslinking can be achieved by reaction of the polysaccharide(s) with the crosslinking agent or by sol-gel reaction of the functionalized polysaccharide by reaction of the polysaccharide with the functionalizing agent.

[0139] Crosslinking is carried out by placing the reaction medium obtained at the end of step c) under conditions allowing crosslinking of the polysaccharide, namely at a pressure P less than or equal to atmospheric pressure and at a temperature T greater than the temperature of the eutectic point of the reaction medium as measured at pressure P and less than the temperature of the freezing point of the reaction medium as measured at pressure P, preferably for a period ranging from 1 week to 17 weeks or from 2 weeks to 17 weeks.

[0140] The pressure P is advantageously equal to atmospheric pressure or less than atmospheric pressure.

[0141] The freezing point temperature of the reaction medium refers to the temperature at which the mixture of the components of the reaction medium, on a macroscopic scale, solidifies, i.e. it becomes non-fluid. Below the freezing point, the mixture is in a frozen state characterized by the coexistence of components in solid and liquid form. The frozen state is maintained up to the eutectic point temperature of the reaction medium.

[0142] The eutectic point temperature of the reaction medium refers to the temperature below which the mixture of the components of the reaction medium passes from a frozen state (coexistence of liquid and solid phases) to a completely solid state, i.e. a state in which all the components of the mixture are in solid form. The freezing point and the eutectic point of a mixture depend on the pressure to which the mixture is subjected, therefore the freezing point and the eutectic point are measured at pressure P.

[0143] The freezing point and eutectic point can be determined by differential scanning calorimetry. This method allows phase transitions to be determined. To do this, the product to be studied is gradually cooled until its phase transitions are observed.

[0144] The temperature T is preferably greater than or equal to -55°C and less than or equal to -5°C, preferably it ranges from -35°C to -10°C. Even more preferably, the temperature T is approximately -20°C.

[0145] The pressure P is preferably atmospheric pressure. "Atmospheric pressure" is the pressure exerted by the air constituting the atmosphere on any surface in contact with it. It varies according to altitude. At an altitude of 0 m, the average atmospheric pressure is 101,325 Pa. Preferably, the pressure P is atmospheric pressure and the temperature T is greater than or equal to -55°C and less than or equal to -5°C, preferably T varies from -35°C to -10°C or is approximately -20°C.

[0146] The crosslinking of the polysaccharide according to step d) can be carried out under vacuum, in particular at a pressure P lower than atmospheric pressure, preferably at a pressure P of between 0.7.10 5 and 0.9.105 Pa (between 0.7 and 0.9 bar), preferably between 0.7.10 5 and 0.8.10 5 Pa (between 0.7 and 0.8 bar).

[0147] Crosslinking step d) is typically carried out for a period of at least 1 hour, preferably at least 3 hours, preferably at least 72 hours, preferably at most 27 weeks. Preferably, crosslinking step d) is carried out for a period of from 1 week to 25 weeks or from 1 to 20 weeks or from 1 to 17 weeks or from 2 to 25 weeks, preferably from 2 to 20 weeks or from 2 to 17 weeks, even more preferably from 3 to 8 weeks or from 4 to 7 weeks and at temperature T, at pressure P.

[0148] Crosslinking using a crosslinking agent

[0149] Crosslinking of the polysaccharide mainly takes place during step d) (it can however begin as early as step c)). This step therefore allows the polysaccharide chains to be crosslinked together. The functional groups of the crosslinking agent react with functional groups present on the polysaccharides so as to link the polysaccharide chains together and crosslink them by forming intermolecular bonds. The crosslinking agent can also react with functional groups present on the same polysaccharide molecule so as to form intramolecular bonds. In particular, the functional groups of the crosslinking agent react with the -OH or -COOH groups, or even -CHO, present on polysaccharides such as hyaluronic acid.Crosslinked polysaccharides comprising at least one crosslinking link between two polysaccharide chains, said crosslinking link being the residue of the crosslinking agent are thus obtained.

[0150] Crosslinking can be carried out in the presence of several crosslinking agents. When crosslinking is carried out in the presence of several crosslinking agents, the crosslinking agents can be added simultaneously or separately over time to the reaction medium. Step d) can thus comprise repeated crosslinking steps. Crosslinking is then carried out in the presence of a total amount of crosslinking agents ranging from 0.1 to 3 moles or from 0.1 to 2 moles or from 0.1 to 1.5 moles or from 0.1 to 1 mole or from 0.1 to 0.8 moles or from 0.1 to 0.5 moles of crosslinking agents (or their salts) per 100 moles of repeating unit of the polysaccharide. The crosslinking conditions, in particular the contents of crosslinking agent, duration and temperatures as well as the weight-average molecular masses (Mw) of the polysaccharide, used are interdependent.

[0151] The lower the crosslinking agent content, the longer the reaction time must be to obtain similar mechanical properties of the resulting gel. In other words, the lower the molar percentage of crosslinking agent, the fewer reactive functions there are in the reaction medium and the lower the probability that 2 groups meet and react together, thus the longer the reaction time must be to allow the functions to react with each other and form crosslinking bonds, and thus obtain a hydrogel with desirable properties.

[0152] In a variant of the process for preparing the hydrogel, the present step d) may be carried out at room temperature (20-25°C) for a period which may be shorter than that described above, for example for a period ranging from 1 hour to 7 days.

[0153] Sol-gel crosslinking

[0154] Functionalization of the polysaccharide

[0155] When crosslinking is carried out by sol-gel reaction, crosslinking reaction d) is carried out by sol-gel reaction of the functionalized polysaccharide, the functionalized polysaccharide being obtained by reaction of the polysaccharide with the functionalizing agent(s). The functionalization of the polysaccharide and the sol-gel reaction may be sequential or at least partly concomitant. The polysaccharide is typically functionalized with at least one molecule of formula Chem. Il as presented above so as to become the bearer of Si-OR groups which will be able to react together and lead to a crosslinked polysaccharide.

[0156] The molecule with the formula Chem. It comprises a single reactive function with respect to the polysaccharide and allows crosslinking only via a sol-gel reaction, it does not present the toxicity of conventional crosslinking agents: the molecule with the formula Chem. It cannot directly crosslink biological molecules (proteins, DNA, etc.). More specifically, the functional group T of the molecule with the formula Chem. It reacts with a functional group present on the polysaccharides in order to functionalize the polysaccharide chains. In particular, the functional group T of the molecule Chem. It thus reacts with an -OH or -COOH group, or a CHO function, present on polysaccharides such as hyaluronic acid.This produces functionalized polysaccharides comprising pendant links on a polysaccharide chain, said pendant links comprising a group -A-Si(R5)(R6)OR10, the group -A-Si(R5)(R6)OR10 coming from the molecule of formula Chem. It can provide biological properties to the hydrogel.

[0157] The solvent is typically water or a mixture comprising water and an organic solvent (e.g. an alcohol, in particular ethanol, or DMSO; typically a mixture comprising at least 90% by weight of water, or at least 95% or at least 98% by weight of water relative to the total weight of the solvent).

[0158] The reaction medium typically comprises from 0.001 to 0.50, preferably from 0.005 to 0.45, in particular from 0.10 to 0.25 mole of molecule of formula Chem. II or a salt thereof per 1 mole of repeating unit of the polysaccharide or from 0.001 to 0.03 mole, preferably from 0.001 to 0.02 mole of molecule of formula Chem. II or a salt thereof per 1 mole of repeating unit of the polysaccharide.

[0159] The mass concentration of polysaccharide in the reaction medium is advantageously between 50 and 300 mg / g of solvent, preferably between 100 and 200 mg / g.

[0160] In certain embodiments, in particular when T is an epoxide, the functionalization is carried out at a pH greater than or equal to 9, or greater than or equal to 10, more advantageously greater than or equal to 12, and in particular at a pH less than 14, for example less than or equal to 13.5. For this, the reaction medium preferably comprises a Bronsted base, more preferably a hydroxide, even more preferably sodium or potassium hydroxide. Advantageously, the reaction medium comprises sodium or potassium hydroxide at a concentration of between 0.10M and 0.30M. In certain embodiments, in particular when T is an amino group, the functionalization is carried out at a pH less than 7, more advantageously greater than or equal to 4.5 and less than 7 or less than or equal to 6.5.For this, the reaction medium preferably comprises a Bronsted acid, more preferably hydrochloric acid, sulfuric acid, or acetic acid.

[0161] In certain embodiments, the functionalization of the polysaccharide is carried out at atmospheric pressure and at a temperature between 4°C and 60°C, preferably between 10°C and 50°C, more preferably between 15°C and 25°C. In these embodiments, the duration of the functionalization reaction can vary from 1 hour to 2 weeks, more particularly from 3 hours to 1 week, even more particularly from 3 hours to 96 hours, for example from 3 hours to 80 hours, in particular from 3 hours to 75 hours.

[0162] In certain embodiments, in particular when the functionalization and the crosslinking of the polysaccharide are concomitant or partly concomitant, the functionalization of the polysaccharide can be, at least partly, carried out at a temperature T higher than the temperature of the eutectic point of the reaction medium as measured at the pressure P and lower than the temperature of the freezing point of the reaction medium as measured at the pressure P. Here, the pressure P is advantageously atmospheric pressure.

[0163] The higher the functionalization temperature, the shorter the functionalization time can be to obtain the same degree of modification by functionalization.

[0164] Sol-gel reaction

[0165] The "sol-gel reaction" consists of forming Si-O-Si bonds from Si-OR groups with R representing a hydrogen atom, an aryl group or an aliphatic hydrocarbon group containing 1 to 6 carbon atoms. This reaction takes place as follows:

[0166] (i) if R is not a hydrogen atom, a step of hydrolysis of at least part of the Si-OR groups to give Si-OH groups; then

[0167] (ii) a step of condensation of the Si-OH groups two by two or of a Si-OH group with a Si-OR group to form Si-O-Si bonds.

[0168] The functionalized polysaccharide is crosslinked by sol-gel reaction to give a hydrogel.

[0169] This step allows the polysaccharide chains to be crosslinked together when they are functionalized with molecules of formula Chem. II. Indeed, during this step, at least some of the Si-OR10 groups and optionally at least some of the SiOR4 groups will react two by two, possibly after hydrolysis of these groups, to form Si-O-Si bonds. This implies that two molecules of formula Chem. II grafted onto polysaccharide chains will react together via their Si-OR10 terminal groups (or even SiOR4 where appropriate) and bond covalently via the formation of a Si-O-Si bond, thus allowing the polysaccharide chains to be bonded together and crosslinked.

[0170] Crosslinked polysaccharides are thus obtained comprising crosslinking links between two polysaccharide chains, said crosslinking links comprising a divalent group -Si- O-Si-,

[0171] The pressure P is advantageously less than or equal to atmospheric pressure.

[0172] The crosslinking of the polysaccharide according to step d) can be carried out under vacuum, in particular at a pressure P lower than atmospheric pressure, preferably at a pressure P of between 0.7.10 5 and 0.9.10 5 Pa (between 0.7 and 0.9 bar), preferably between 0.7.10 5 and 0.8.10 5 Pa (between 0.7 and 0.8 bar).

[0173] The pressure P is advantageously atmospheric pressure.

[0174] In some embodiments, the polysaccharide may be crosslinked by a crosslinking agent and by sol-gel reaction (using a functionalizing agent). The reactions may be successive or concomitant.

[0175] Additional steps

[0176] The process for preparing the hydrogel according to the invention may comprise one or more additional steps, such as, for example, steps of adding one or more additional components, purification, sterilization, sieving, swelling and / or conditioning.

[0177] The method for preparing the hydrogel according to the invention may comprise a step of adding at least one additional component. The additional component may be chosen from lubricating agents; cosmetic active ingredients such as antioxidants, coenzymes, amino acids, vitamins, minerals, and nucleic acids; therapeutic active ingredients such as anesthetics, antibiotics, antifungals and adrenaline and its derivatives, and mixtures thereof. The additional components may be as described above.

[0178] The method for preparing the hydrogel according to the invention may comprise at least one purification step. The purification may be carried out by dialysis. The method for preparing the hydrogel according to the invention may comprise a step of sterilizing the hydrogel. The sterilization is preferably carried out by heat. The sterilization is generally carried out by increasing the temperature of the sterilization medium to a temperature called the "plateau temperature", which is maintained for a determined period of time called the "plateau time". The sterilization is preferably carried out at a plateau temperature ranging from 121°C to 135°C, preferably with a plateau time ranging from 1 minute to 20 minutes with FO > 15. The sterilizing value FO corresponds to the time required, in minutes, at 121°C, to inactivate 90% of the population of microorganisms present in the product to be sterilized.Alternatively, sterilization can be carried out by gamma ray, UV radiation or ethylene oxide.

[0179] The method may comprise a step of sieving the hydrogel, more particularly with a sieve with a porosity of between 50 and 2000 μm. This sieving step makes it possible to obtain a more homogeneous hydrogel with the most constant extrusion force possible, i.e., the most regular possible. A person skilled in the art knows how to select a sieve with a suitable pore size depending on the mechanical properties of the hydrogel being prepared.

[0180] The method may comprise a hydrogel swelling step. During the hydrogel swelling step, the polysaccharide concentration of the hydrogel is adapted. In particular, a solvent is added, for example, water, a phosphate buffer, water for injection. More particularly, the added solvent has a pH around physiological pH (6.8-7.8). The polysaccharide concentration obtained following the swelling step advantageously varies from 1 mg / g to 50 mg / g of hydrogel, more advantageously from 5 mg / g to 35 mg / g of hydrogel, even more advantageously from 10 mg / g to 30 mg / g of hydrogel.

[0181] The method may comprise a step of conditioning the hydrogel, for example in an injection device.

[0182] Where appropriate, the step of adding one or more additional components preferably takes place after the purification step.

[0183] Where appropriate, the step of adding one or more additional components preferably takes place before the sterilization step.

[0184] In particular, the step of adding one or more additional components may also comprise the addition of at least one therapeutic active ingredient, or at least one cosmetic active ingredient, or their mixture. When at least one therapeutic active ingredient and / or at least one cosmetic active ingredient is added, the step of adding one or more additional components preferably takes place after step d).

[0185] Where appropriate, the purification step preferably takes place after the step of adding one or more additional components.

[0186] Where applicable, the purification step preferably takes place before the sterilization step.

[0187] Where appropriate, the purification step preferably takes place before the sieving step.

[0188] The sterilization step is preferably carried out after steps a) to d) and any additional steps. In particular, the hydrogel is sterilized after being packaged in its injection device and the packaging of the hydrogel takes place following all steps of the process and before sterilization.

[0189] Applications

[0190] The hydrogel according to the invention can be injected using any of the methods known to those skilled in the art. In particular, a hydrogel according to the invention can be administered by means of an injection device suitable for intra-epidermal and / or intradermal and / or subcutaneous and / or supra-periosteal injection. The injection device can in particular be chosen from a syringe, a set of micro-syringes, a thread, a laser or hydraulic device, an injection gun, a needle-free injection device, or a micro-needle roller.

[0191] The present invention also relates to an injection device comprising a hydrogel according to the invention.

[0192] The hydrogel according to the invention is preferably injected subcutaneously.

[0193] The hydrogel according to the present invention may particularly relate to deep applications.

[0194] The hydrogel according to the invention may have therapeutic and / or cosmetic applications.

[0195] In certain embodiments, the present invention also relates to a hydrogel for its non-therapeutic use in filling and / or replacing tissues, in particular soft tissues, in particular by injecting the hydrogel into the tissue.

[0196] In the cosmetic field, the hydrogel according to the invention can be particularly useful for compensating for losses of tissue volume due to aging.

[0197] The hydrogel according to the present invention can be used in the prevention and / or cosmetic treatment of an alteration of the surface appearance of the skin. For example, the hydrogel according to the invention can be used in the cosmetic field to prevent and / or treat the alteration of the viscoelastic or biomechanical properties of the skin; to fill volume defects of the skin, in particular to fill wrinkles, fine lines and scars; to reduce nasolabial folds and bitterness folds; to increase the volume of the cheekbones, chin or lips; to restore the volumes of the face, in particular the cheeks, temples, the oval of the face, and the area around the eyes; to reduce the appearance of wrinkles and fine lines.

[0198] The present invention also relates to the cosmetic use of a hydrogel as described above for filling tissues, in particular soft tissues, in particular to compensate for losses of tissue volume due to aging.

[0199] EXAMPLES

[0200] 1. Materials

[0201] - 3MDa uncrosslinked sodium hyaluronate,

[0202] - BDDE,

[0203] - NaOH 0.25M,

[0204] - 1 M HCl,

[0205] - Phosphate buffer,

[0206] - Lidocaine hydrochloride,

[0207] - Three-dimensional agitator,

[0208] - DHR-2 Rheometer - TA Instrument ®,

[0209] - Dynamometer and test bench,

[0210] - Homogenizer Paddle mill,

[0211] - Sterile polyethylene bag.

[0212] 2. Methods

[0213] A hydrogel according to the invention (Gel A) is compared with commercial volumizing hydrogels Juvederm Voluma® - 20 mg / g (Allergan®), RHA 4 - 23 mg / g (Teoxane®), Belotero® Volume - 26 mg / g (Merz Pharma®). All four hydrogels compared are sterile.

[0214] Unless otherwise stated, the steps described below are carried out at room temperature (21°C). Preparation of the hydrogels according to the invention:

[0215] BDDE and sodium hyaluronate (3 MDa, 120 mg / g) were dissolved in a 0.25 M aqueous sodium hydroxide solution in a sterile bag. The mixture was then homogenized in a paddle mill until the sodium hyaluronate was completely dissolved (i.e., until a transparent and perfectly homogeneous product (i.e., absence of agglomerate) was obtained). The resulting mixture was maintained at atmospheric pressure and placed at -20 °C for the duration shown in Table 1. The pH of the resulting mixture was approximately 13.

[0216] A 1 N HCl solution was then added to the sterile bag until a pH of 7.3 ± 0.5 was obtained. The resulting mixture was diluted with PBS phosphate buffer to a concentration of 25 mg of hyaluronic acid per gram of product for Gel A. The resulting mixture was homogenized for 24 h using a three-dimensional stirrer. The mixture was dialyzed.

[0217] A solution of high molecular weight uncrosslinked sodium hyaluronate was added as a lubricant.

[0218] An aqueous solution of lidocaine hydrochloride is added to obtain 0.3% by weight of lidocaine hydrochloride relative to the weight of the resulting product.

[0219] The product thus obtained was sieved to the order of a micron and then packaged in a syringe.

[0220] Finally, the product was sterilized in an autoclave (plate temperature between 121°C and 135°C with FO > 15).

[0221] The crosslinking conditions of the hydrogel according to the invention are presented in Table 1 below.

[0222] Table 1: Hydrogel crosslinking conditions

[0223] The force of 2 Newton applied in the method for determining the projection index is included in the LVER interval of the 4 hydrogels to be evaluated. The projection index values ​​of these 4 hydrogels are measured at 25°C using a DHR2 rheometer equipped with a geometry of parallel flat plates (diameter 40mm, anodized aluminum, TA instruments®) associated with TRIOS software (TA Instruments®). 1 gram of hydrogel is applied between the plates and the gap between these plates corresponds to an initial thickness of 700 μm. A compression force of 2 Newton is then applied for 1 hour and the evolution of the variation in the thickness of the hydrogel is acquired. The parameter c is calculated using the OriginPro® 2019 software version 9.6.0.172.

[0224] We apply the generalized Maxwell model, which gives the evolution of the thickness of the hydrogel as a function of time t by the formula: where d~ is the limit thickness obtained at equilibrium, A a constant, Ti a relaxation parameter, the number of members i of the equation equal to 2.

[0225] The results are obtained with the presence of two exponential terms:

[0226] The projection index values ​​are then calculated as follows: d0 being the initial thickness, i.e. 700 pm and d œ being obtained through the application of the generalized Maxwell model.

[0227] The MOD of each of the hydrogels are measured by 1H NMR. The hydrogel was precipitated in isopropanol and dried for 6 hours under vacuum. The resulting hyaluronic acid residues were dissolved at 10 mg / mL in D2O. 50 pL hyaluronidase (Type VI-S from bovine testes, 3 kU / mL in D2O) was added to degrade the gels for 18 hours at 37°C. The analysis was performed on a Bruker Avance 400 MHz spectrometer. The MOD was determined according to the following equation:

[0228] MOD (%) = (I 5H1 - 6 - 7 / 4) / (I 5H2 -^ 2 1 / 3)-100

[0229] The minimum quantification limit of MOD is 1%.

[0230] The results are presented in Table 2 below.

[0231] Table 2: Results

[0232] The hydrogel according to the invention, Gel A, has a Pldx projection index greater than 80% demonstrating a strong capacity to sustainably maintain its thickness in the layers of the skin.

[0233] Commercial hydrogels have a projection index of less than 80%, they have a lower capacity to maintain their thickness under a force of 2 N than the hydrogel according to the invention. In addition, the calculated MOD / PIdx is much lower for Gel A compared to commercial hydrogels demonstrating a strong capacity to sustainably maintain its thickness under a force of 2 N while having a low degree of modification.

Claims

DEMANDS 1. Crosslinked polysaccharide-based hydrogel having a projection index PIdx greater than or equal to 80%, the projection index being determined according to a method comprising the following steps: 1) subject a bolus of one gram of hydrogel to a fixed constant compressive force F of 2 newtons, said hydrogel being previously placed between two circular pressure plates of a rheometer and having an initial thickness dO of 700 pm given by the distance between the two plates, the two plates being parallel and having a diameter of 40 mm; 2) acquire at 25°C the evolution of the variation in thickness of the hydrogel thus compressed for 1 hour; 3) parameterize a generalized Maxwell model approximating the evolution observed from the acquisition performed in step 2), said model expressing the hydrogel thickness as a function of time d ge i(t) and corresponding to the following equation 1: (equation 1) where d~ is the limiting thickness obtained at equilibrium, Ai a constant, Ti a relaxation parameter, the number of members i of the equation being equal to 2; 4) determine from the model a limiting thickness d~ towards which the hydrogel tends to evolve over time; and 5) Generate the projection index PIdx from the following equation 2: 100 (equation 2).

2. Hydrogel according to claim 1 wherein the hydrogel has a degree of modification (MOD) / PIdx ratio equal to or less than 0.050, preferably equal to or less than 0.040, more preferably equal to or less than 0.030, even more preferably equal to or less than 0.

015.

3. Hydrogel according to claim 1 or 2 having a projection index PIdx greater than or equal to 82%, preferably greater than or equal to 85%, more preferably greater than or equal to 90%.

4. Hydrogel according to any one of the preceding claims wherein the polysaccharide has a degree of modification (MOD) of less than or equal to 3%, preferably less than or equal to 2%, more preferably less than or equal to 1%.

5. Hydrogel according to any one of the preceding claims further comprising a non-crosslinked polysaccharide, preferably non-crosslinked hyaluronic acid, non-crosslinked heparosan or a mixture thereof.

6. Hydrogel according to any one of the preceding claims wherein the cross-linked polysaccharide represents from 0.1 to 5% by weight, preferably from 1 to 3% by weight, relative to the total weight of the hydrogel.

7. Hydrogel according to any one of claims 1 to 6 comprising a total polysaccharide concentration in the hydrogel ranging from 1 mg / g to 50 mg / g of hydrogel, preferably from 5 mg / g to 35 mg / g of hydrogel, even more preferably from 10 mg / g to 30 mg / g of hydrogel.

8. Hydrogel according to any one of the preceding claims wherein the crosslinked polysaccharide is prepared by reacting the polysaccharide with a crosslinking agent selected from the group consisting of 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxy-octane, poly(ethylene glycol) diglycidyl ether (PEGDG), 1,2-bis(2,3-epoxypropoxy)ethane (EGDGE), and mixtures thereof.

9. Hydrogel according to any one of claims 1 to 7, wherein the cross-linked polysaccharide is prepared by sol-gel reaction of a polysaccharide functionalized with a functionalizing agent of formula Chem. II: Chem. It or a salt thereof, in which: T represents an isocyanate, amino, epoxide, carboxyl, N-succinimidyloxycarbonyl, N-sulfosuccinimidyloxycarbonyl, halocarbonyl, isothiocyanate, vinyl, formyl, hydroxyl, sulfhydryl, hydrazino, acylhydrazino, aminoxy, carbodiimide, or acid anhydride residue group; - A represents a chemical bond or a spacer group; - R5 and R6, whether identical or different, represent a hydrogen atom; a halogen atom; an -OR4 group with R4 representing a hydrogen atom, an aryl group, or an aliphatic hydrocarbon group comprising 1 to 6 carbon atoms; an aryl; or an aliphatic hydrocarbon group comprising 1 to 6 carbon atoms, possibly substituted by one or more groups selected from a halogen atom, an aryl, and a hydroxyl; and - R10 represents a hydrogen atom, an aryl group or an aliphatic hydrocarbon group containing 1 to 6 carbon atoms.

10. Hydrogel according to any one of claims 1 to 8 prepared by a process comprising the following steps: a) providing at least one polysaccharide or a salt thereof; b) providing at least one crosslinking agent or a salt thereof; c) preparing a crosslinking reaction medium comprising the polysaccharide(s), the crosslinking agent(s), and a solvent, the total amount of crosslinking agent being from 0.001 to 0.03 mol per 1 mol of repeating unit of the polysaccharide, preferably with the preparation of the reaction medium not exceeding 5 hours; and (d) place the reaction medium obtained at the end of step (c) at a pressure P less than or equal to atmospheric pressure and at a temperature T greater than the eutectic point temperature of the reaction medium as measured at pressure P and less than the freezing point temperature of the reaction medium as measured at pressure T. P, preferably for a period ranging from 1 week to 17 weeks.

11. Hydrogel according to any one of claims 1 to 7 or 9 prepared by a process comprising the following steps: a) providing at least one polysaccharide or a salt thereof; b) providing at least one functionalizing agent or a salt thereof as described in claim 9; (c) prepare a crosslinking reaction medium comprising the polysaccharide(s), the functionalizing agent(s) and a solvent, the total amount of functionalizing agent ranging from 0.001 to 0.50 mole per 1 mole of repeating unit of the polysaccharide, preferably the duration of the preparation step of the reaction medium not exceeding 5 hours; and (d) place the reaction medium obtained at the end of step (c) at a pressure P less than or equal to atmospheric pressure and at a temperature T greater than the eutectic point temperature of the reaction medium as measured at pressure P and less than the freezing point temperature of the reaction medium as measured at pressure P, preferably for a period ranging from 1 week to 17 weeks.

12. Hydrogel according to any one of the preceding claims wherein the cross-linked polysaccharide is hyaluronic acid, a salt of hyaluronic acid or mixtures thereof.

13. Hydrogel according to any one of the preceding claims in sterile form.

14. Hydrogel according to any one of the preceding claims in injectable form.

15. Hydrogel according to any one of the preceding claims further comprising an anesthetic agent.

16. Cosmetic use of a hydrogel according to any one of the preceding claims to prevent and / or treat alterations in the viscoelastic or biomechanical properties of the skin; to fill wrinkles, fine lines and scars; to reduce nasolabial folds and marionette lines; to increase the volume of cheekbones, chin or lips or to reduce the appearance of wrinkles and fine lines.

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